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MicroRNA-132-3p represses Smad5 in MC3T3-E1 osteoblastic cells under cyclic tensile stress

Molecular and Cellular Biochemistry volume 458, pages 143–157 (2019)Cite this article

Abstract

MicroRNAs (miRNAs) regulate osteogenic differentiation of bone cells, which has applications in orthodontics. Here we evaluated the miRNA expression profile of MC3T3-E1 osteoblasts under cyclic tensile stress with chip technology and found that miR-132-3p was up-regulated by 12% cyclic tensile stress. Alkaline phosphatase activity and osteocalcin expression in MC3T3-E1 cells were decreased under these conditions. Smad2 and Smad5 were identified as potential target genes of miR-132-3p. Native and phosphorylated Smad2 and Smad5 expression was negatively correlated with miR-132-3p levels in the cells under cyclic stretch; however, only Smad5 protein level was reduced upon miR-132-3p overexpression. The luciferase reporter assay confirmed a direct interaction between miR-132-3p and Smad5. Thus, miR-132-3p maybe regulates osteoblast differentiation via Smad5 in response to cyclic tensile stress.

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References

  1. Lian JB, Stein GS, van Wijnen AJ, Stein JL, Hassan MQ, Gaur T, Zhang Y (2012) MicroRNA control of bone formation and homeostasis. Nat Rev Endocrinol 8:212–227. https://doi.org/10.1038/nrendo.2011.234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Liu L, Liu M, Li R, Liu H, Du L, Chen H, Zhang Y, Zhang S, Liu D (2017) Microrna-503-5p inhibits stretch-induced osteogenic differentiation and bone formation. Cell Biol Int 41:112–123. https://doi.org/10.1002/cbin.10704

    Article  CAS  PubMed  Google Scholar 

  3. Chang M, Lin H, Fu H, Wang B, Han G, Fan M (2017) Microrna-195-5p regulates osteogenic differentiation of periodontal ligament cells under mechanical loading. J Cell Physiol 232:3762–3774. https://doi.org/10.1002/jcp.25856

    Article  CAS  PubMed  Google Scholar 

  4. Chen N, Sui BD, Hu CH, Cao J, Zheng CX, Hou R, Yang ZK, Zhao P, Chen Q, Yang QJ et al (2016) Microrna-21 contributes to orthodontic tooth movement. J Dent Res 95:1425–1433. https://doi.org/10.1177/0022034516657043

    Article  CAS  PubMed  Google Scholar 

  5. Wu Y, Yang Y, Yang P, Gu Y, Zhao Z, Tan L, Zhao L, Tang T, Li Y (2013) The osteogenic differentiation of PDLSCs is mediated through MEK/ERK and p38 MAPK signalling under hypoxia. Arch Oral Biol 58:1357–1368. https://doi.org/10.1016/j.archoralbio.2013.03.011

    Article  CAS  PubMed  Google Scholar 

  6. Papachroni KK, Karatzas DN, Papavassiliou KA, Basdra EK, Papavassiliou AG (2009) Mechanotransduction in osteoblast regulation and bone disease. Trends Mol Med 15:208–216. https://doi.org/10.1016/j.molmed.2009.03.001

    Article  CAS  PubMed  Google Scholar 

  7. Wu M, Chen G, Li YP (2016) TGF-β and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res 4:16009. https://doi.org/10.1038/boneres.2016.9

    Article  PubMed  PubMed Central  Google Scholar 

  8. Rahman MS, Akhtar N, Jamil HM, Banik RS, Asaduzzaman SM (2015) TGF-β/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation. Bone Res 3:15005. https://doi.org/10.1038/boneres.2015.5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yao W, Pan Z, Du X, Zhang J, Li Q (2018) miR-181b-induced SMAD7 downregulation controls granulosa cell apoptosis through TGF-β signaling by interacting with the TGFBR1 promoter. J Cell Physiol 233:6807–6821. https://doi.org/10.1002/jcp.26431

    Article  CAS  PubMed  Google Scholar 

  10. Jin W, Chen F, Wang K, Song Y, Fei X, Wu B (2018) miR-15a/miR-16 cluster inhibits invasion of prostate cancer cells by suppressing TGF-β signaling pathway. Biomed Pharmacother 104:637–644. https://doi.org/10.1016/j.biopha.2018.05.041

    Article  CAS  PubMed  Google Scholar 

  11. Wei F, Yang S, Guo Q, Zhang X, Ren D, Lv T, Xu X (2017) MicroRNA-21 regulates osteogenic differentiation of periodontal ligament stem cells by targeting Smad5. Sci Rep 7:16608. https://doi.org/10.1038/s41598-017-16720-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Song Z, Chen LL, Wang RF, Qin W, Huang SH, Guo J, Lin ZM, Tian YG (2017) MicroRNA-135b inhibits odontoblast-like differentiation of human dental pulp cells by regulating Smad5 and Smad4. Int Endod J 50:685–693. https://doi.org/10.1111/iej.12678

    Article  CAS  PubMed  Google Scholar 

  13. Liu M, Dai J, Lin Y, Yang L, Dong H, Li Y, Ding Y, Duan Y (2012) Effect of the cyclic stretch on the expression of osteogenesis genes in human periodontal ligament cells. Gene 491:187–193. https://doi.org/10.1016/j.gene.2011.09.031

    Article  CAS  PubMed  Google Scholar 

  14. Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ (1992) Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res 7:683–692. https://doi.org/10.1002/jbmr.5650070613

    Article  CAS  PubMed  Google Scholar 

  15. Wang ZH, Zhang QS, Duan YL, Zhang JL, Li GF, Zheng DL (2015) TGF-β induced miR-132 enhances the activation of TGF-β signaling through inhibiting SMAD7 expression in glioma cells. Biochem Biophys Res Commun 463:187–192. https://doi.org/10.1016/j.bbrc.2015.05.001

    Article  CAS  PubMed  Google Scholar 

  16. Zhang Q, Ye H, Xiang F, Song LJ, Zhou LL, Cai PC, Zhang JC, Yu F, Shi HZ, Su Y et al (2017) Mir-18a-5p inhibits sub-pleural pulmonary fibrosis by targeting TGF-β receptor II. Mol Ther 25:728–738. https://doi.org/10.1016/j.ymthe.2016.12.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang Q, Wang Z, Chu L, Li X, Kan P, Xin X, Zhu Y, Yang P (2015) The effects and molecular mechanisms of MiR-106a in multidrug resistance reversal in human glioma U87/DDP and U251/G cell lines. PLoS ONE 10:e0125473. https://doi.org/10.1371/journal.pone.0125473

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang Y, Du J, Niu X, Fu N, Wang R, Zhang Y, Zhao S, Sun D, Nan Y (2017) MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2. Cell Death Dis 8:e2792. https://doi.org/10.1038/cddis.2017.10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mohamed JS, Hajira A, Lopez ML, Boriek AM (2015) Genome-wide mechanosensitive microRNA (MechanomiR) screen uncovers dysregulation of their regulatory networks in the mdm mouse model of muscular dystrophy. J Biol Chem 290:24986–25011. https://doi.org/10.1074/jbc.M115.659375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Park SO, Kumar M, Gupta S (2012) TGF-β and iron differently alter HBV replication in human hepatocytes through TGF-β/BMP signaling and cellular microRNA expression. PLoS ONE 7:e39276. https://doi.org/10.1371/journal.pone.0039276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lin SZ, Xu JB, Ji X, Chen H, Xu HT, Hu P, Chen L, Guo JQ, Chen MY, Lu D et al (2015) Emodin inhibits angiogenesis in pancreatic cancer by regulating the transforming growth factor-β/drosophila mothers against decapentaplegic pathway and angiogenesis-associated microRNAs. Mol Med Rep 12:5865–5871. https://doi.org/10.3892/mmr.2015.4158

    Article  CAS  PubMed  Google Scholar 

  22. Ye EA, Liu L, Steinle JJ (2017) miR-15a/16 inhibits TGF-beta3/VEGF signaling and increases retinal endothelial cell barrier proteins. Vision Res 139:23–29. https://doi.org/10.1016/j.visres.2017.07.007

    Article  PubMed  PubMed Central  Google Scholar 

  23. Wei B, Wei W, Zhao B, Guo X, Liu S (2017) Long non-coding RNA HOTAIR inhibits miR-17-5p to regulate osteogenic differentiation and proliferation in non-traumatic osteonecrosis of femoral head. PLoS ONE 12:e0169097. https://doi.org/10.1371/journal.pone.0169097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cheng D, Zhao S, Tang H, Zhang D, Sun H, Yu F, Jiang W, Yue B, Wang J, Zhang M et al (2016) MicroRNA-20a-5p promotes colorectal cancer invasion and metastasis by downregulating Smad4. Oncotarget 7:45199–45213. https://doi.org/10.18632/oncotarget.9900

    Article  PubMed  PubMed Central  Google Scholar 

  25. Feller L, Khammissa RAG, Schechter I, Thomadakis G, Fourie J, Lemmer J (2015) Biological events in periodontal ligament and alveolar bone associated with application of orthodontic forces. ScientificWorldJournal 2015:1–7. https://doi.org/10.1155/2015/876509

    CAS  Article  Google Scholar 

  26. Feller L, Khammissa RAG, Schechter I, Moodley A, Thomadakis G, Lemmer J (2015) Periodontal biological events associated with orthodontic tooth movement: the biomechanics of the cytoskeleton and the extracellular matrix. ScientificWorldJournal 2015:894123. https://doi.org/10.1155/2015/894123

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Jackson RA, Kumarasuriyar A, Nurcombe V, Cool SM (2006) Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells. J Cell Physiol 209:894–904. https://doi.org/10.1002/jcp.20779

    Article  CAS  PubMed  Google Scholar 

  28. Chen X, Guo J, Yuan Y, Zhang L, Wu J, Sun Z, Chen B, Zou J (2016) The effect of mechanical strain on the differentiation of MC3T3-E1 osteoblasts and the Wnt signaling pathway. Chin J Osteoporos. https://doi.org/10.3969/j.issn.1006-7108.2016.01.002 (in Chinese)

    Article  Google Scholar 

  29. Chung E, Rylander MN (2011) Response of a preosteoblastic cell line to cyclic tensile stress conditioning and growth factors for bone tissue engineering. Tissue Eng Part A 18:397–410. https://doi.org/10.1089/ten.TEA.2010.0414

    Article  PubMed  PubMed Central  Google Scholar 

  30. Wall ME, Weinhold PS, Siu T, Brown TD, Banes AJ (2007) Comparison of cellular strain with applied substrate strain in vitro. J Biomech 40:173–181. https://doi.org/10.1016/j.jbiomech.2005.10.032

    Article  PubMed  Google Scholar 

  31. Park JS, Chu JS, Cheng C, Chen F, Chen D, Li S (2004) Differential effects of equiaxial and uniaxial strain onmesenchymal stem cells. Biotechnol Bioeng 88:359–368. https://doi.org/10.1002/bit.20250

    Article  CAS  PubMed  Google Scholar 

  32. Sang S, Zhang Z, Qin S, Li C, Dong Y (2017) MicroRNA-16-5p inhibits osteoclastogenesis in giant cell tumor of bone. Biomed Res Int 2017:3173547. https://doi.org/10.1155/2017/3173547

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Haenisch S, Zhao Y, Chhibber A, Kaiboriboon K, Do LV, Vogelgesang S, Barbaro NM, Alldredge BK, Lowenstein DH, Cascorbi I et al (2015) SOX11 identified by target gene evaluation of miRNAs differentially expressed in focal and non-focal brain tissue of therapy-resistant epilepsy patients. Neurobiol Dis 77:127–140. https://doi.org/10.1016/j.nbd.2015.02.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kim HR, Hwang SJ, Shin CH, Choi KH, Ohn T, Kim HH (2017) SRSF3-regulated miR-132/212 controls cell migration and invasion by targeting YAP1. Exp Cell Res 358:161–170. https://doi.org/10.1016/j.yexcr.2017.06.009

    Article  CAS  PubMed  Google Scholar 

  35. Wen Y, Han J, Chen J, Dong J, Xia Y, Liu J, Jiang Y, Dai J, Lu J, Jin G et al (2015) Plasma mi RNAs as early biomarkers for detecting hepatocellular carcinoma. Int J Cancer 137:1679–1690. https://doi.org/10.1002/ijc.29544

    Article  CAS  PubMed  Google Scholar 

  36. Chen Z, Zhang Y, Liang C, Chen L, Zhang G, Qian A (2017) Mechanosensitive miRNAs and bone formation. Int J Mol Sci 18:1684. https://doi.org/10.3390/ijms18081684

    Article  CAS  PubMed Central  Google Scholar 

  37. Qu X, Chen Z, Fan D, Sun C, Zeng Y (2016) MiR-132-3p regulates the osteogenic differentiation of thoracic ligamentum flavum cells by inhibiting multiple osteogenesis-related genes. Int J Mol Sci 17:1370. https://doi.org/10.3390/ijms17081370

    Article  CAS  PubMed Central  Google Scholar 

  38. Hu Z, Wang Y, Sun Z, Wang H, Zhou H, Zhang L, Zhang S, Cao X (2015) miRNA-132-3p inhibits osteoblast differentiation by targeting Ep300 in simulated microgravity. Sci Rep 5:18655. https://doi.org/10.1038/srep18655

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gong K, Qu B, Liao D, Liu D, Wang C, Zhou J, Pan X (2016) MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor β/δ-dependent manner. Biochem Biophys Res Commun 478:260–267. https://doi.org/10.1016/j.bbrc.2016.07.057

    Article  CAS  PubMed  Google Scholar 

  40. Qi L, Zhang Y (2014) The microRNA 132 regulates fluid shear stress-induced differentiation in periodontal ligament cells through mTOR signaling pathway. Cell Physiol Biochem 33:433–445. https://doi.org/10.1159/000358624

    Article  CAS  PubMed  Google Scholar 

  41. Heldin CH, Miyazono K, Ten Dijke P (1997) TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature 390:465–471. https://doi.org/10.1038/37284

    Article  CAS  PubMed  Google Scholar 

  42. Hata A, Chen YG (2016) TGF-β signaling from receptors to Smads. Cold Spring Harb Perspect Biol 8:a022061. https://doi.org/10.1101/cshperspect.a022061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Massagué J (1998) TGF-β signal transduction. Annu Rev Biochem 67:753–791. https://doi.org/10.1146/annurev.biochem.67.1.753

    Article  PubMed  Google Scholar 

  44. Lee KS, Kim HJ, Li QL, Chi XZ, Ueta C, Komori T, Wozney JM, Kim EG, Choi JY, Ryoo HM et al (2000) Runx2 is a common target of transforming growth factor β1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol 20:8783–8792

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Chen G, Deng C, Li YP (2012) TGF-β and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci 8:272–288. https://doi.org/10.7150/ijbs.2929

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Fujii M, Takeda K, Imamura T, Aoki H, Sampath TK, Enomoto S, Kawabata M, Kato M, Ichijo H, Miyazono K (1999) Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation. Mol Biol Cell 10:3801–3813. https://doi.org/10.1091/mbc.10.11.3801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Yoo HS, Chung KH, Lee KJ, Kim DH, An JH (2017) Melanin extract from Gallus gallus domesticus promotes proliferation and differentiation of osteoblastic MG-63 cells via bone morphogenetic protein-2 signaling. Nutr Res Pract 11:190–197. https://doi.org/10.4162/nrp.2017.11.3.190

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Jun JH, Yoon WJ, Seo SB, Woo KM, Kim GS, Ryoo HM, Baek JH (2010) BMP2-activated Erk/MAP kinase stabilizes Runx2 by increasing p300 levels and histone acetyltransferase activity. J Biol Chem 285:36410–36419. https://doi.org/10.1074/jbc.M110.142307

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Nishimura R, Kato Y, Chen D, Harris SE, Mundy GR, Yoneda T (1998) Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12. J Biol Chem 273:1872–1879

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the natural science foundation of China (81400566, 81302220), the natural science foundation of Shanxi Province (2014011027-2, 2014021037-1), the Doctoral Startup Research Fund of Shanxi Medical University (B03201204), and sponsored by the Fund for Shanxi “1331 Project” Key Subjects Construction. We would like to thank Editage http://www.editage.cn for English language editing.

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  1. MingYan Liu and Fen Sun contributed equally to this work.

Affiliations

  1. Department of Orthodontics, Dental Hospital, Shanxi Medical University, Taiyuan, China

    MingYan Liu, Fen Sun & YunXia Feng

  2. Suzhou Dental Doctor Outpatient Department Co. LTD, Suzhou, China

    MingYan Liu

  3. School of Basic Medical Science, Shanxi Medical University, 56 Xinjian Road, Ying Ze, Taiyuan, 030001, China

    XinYi Sun & Ming Liu

  4. Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, 15 Changle Xi Road, Xi’an, China

    Juan Li

  5. Aoji Bio-Tech Co., Ltd, Shanghai, China

    Qiang Fan

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  1. MingYan Liu
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Liu, M., Sun, F., Feng, Y. et al. MicroRNA-132-3p represses Smad5 in MC3T3-E1 osteoblastic cells under cyclic tensile stress. Mol Cell Biochem 458, 143–157 (2019). https://doi.org/10.1007/s11010-019-03538-3

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Keywords

  • MicroRNAs
  • MicroRNA-132-3p
  • Cyclic stretch
  • Smad5